(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl)-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid hydrates pharmaceutical uses thereof

ABSTRACT

The present invention relates to (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid hydrates compounds, preparing methods thereof, pharmaceutical compositions containing said compounds and preparing methods thereof, and the clinical uses of said compounds as neuramidinase inhibitors for anti-influenza.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/673,355, filed Feb. 12, 2010, which a U.S national stage applicationof PCT International Patent Application No. PCT/CN2008/001459, filedAug. 13, 2008, each of which is incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrates compounds, preparing methods thereof, pharmaceuticalcompositions comprising said compounds and use of the same foranti-influenza.

BACKGROUND ART

Suddenly occurring new-type viral infectious disease is a type of highlyinfectious and harmful disease resulted from self-evolution orself-variation of virus. Since its pathogen has an entirely newbiological structure, the disease is generally difficult to prevent andtreat at its outbreak, and thus causes a very serious hazard. Influenzavirus, due to its high variability and the susceptibility of human bodythereto, becomes an important source for suddenly occurring new-typeviral infectious disease. At regular intervals, new-type influenza willbecome widely pandemic in humankind. In recent 100 years, there havebeen at least four times of outbreak of influenza, among which theinfluenza pandemic in 1918 killed almost twenty millions of peoplearound the world. At present, H5N1 type high pathogenic avian influenzais pervading in poultry, and this influenza, along with thediscontinuous variation in itself, will necessarily become a new type ofinfluenza that can infect humankind. As the threat of an outbreak ofinfluenza is drawing near, it is a very important means to develop intime a high-efficiency drug for dealing with the prevalence of suddenlyoccurring new-type viral infectious disease led by new-type influenza.

Neuraminidase (NA) is a key protein that promotes the separation anddiffusion of influenza virus particles as newly formed from infectedcells. Among the three types of proteins (hemagglutinin, HA,neuraminidase, NA and nonstructural protein NS1) that are the easiestvariable on the surface of influenza virus, NA is relatively stable, andin particular, the amino acid sequences thereof that constitute activesites are highly conservative in all influenza A and B viruses.Therefore, the development of an NA inhibitor is the optimal choice fordealing with unknown influenza virus. As far as the drugs for preventingand treating new type of influenza are concerned, NA inhibitorsrepresented by oseltamivir and zanamivir are regarded as potentialeffective drugs. However, the two drugs both have certain limitations.Both of them are oral preparations, are unfavorable for treating highrisk patients, and are also inconvenient to take by susceptiblepopulation such as elderly person and children, so their effects ofprevention and treatment are greatly limited. As for other serious viralinfectious diseases, there aren't effective drugs to prevent and treatthem as well, and the expense for treating them is high. Moreover, thelarge and wide use of a single type of drug easily leads to theoccurrence of drug-resistant virus stains, so that the drug preventionand treatment system will lose its protection ability. Therefore, it isnecessary to continuously search for a new therapeutic route to therebydevelop a drug having good therapeutic effect and low toxic-side effect.

(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid [RWJ270201, BCX1812, JNJ2, peramivir] is a type of cyclopentanecompound that is completely different in structure from oseltamivir andzanamivir, and can selectively inhibit NA of influenza A and B viruses.In vitro activity study showed that RWJ270201 could selectively inhibitin vitro the activity of NA of 7 influenza A virus stains (IC50=0.1-1.4nM), and the activity of NA of 4 influenza B virus strains (IC50=0.6-11nM) (Bania S., Parker C. D., Ananth S. L., et al, Comparison of theAnti-influenza Virus Activity of RWJ270201 against Clinical Isolates ofInfluenza Virus and Neuraminidase Inhibitor-Resistant Variants,Antimicrob Agents Chemother, 2001, 45(12), 3403-3408). The activity ofRWJ270201 was equivalent to or higher than that of oseltamivir andzanamivir. In vivo activity study showed that RWJ270201 could markedlyreduce the death rate of mice that were infected with influenza virusstrain A/HongKong/156/97 (H5N1), resulting in a survival rate of up to70% in the group of 0.1 mg/kg/day, and a survival rate of up to 100% inthe group of 10 mg/kg/day, and could markedly reduce virus titer in lungtissue, and prevent the diffusion of virus to brain tissue (Govorkova E.A., Leneva I. A., Goloubeva O. G., et al., Comparison of Efficiencies ofRWJ270201, Zanamivir, and Oseltamivir against H5N1, H9N2, and OtherAvian Influenza Viruses, Antimicrob Agents Chemother, 2001, 45(10),2723-2732). RWJ270201 could also inhibit lung consolidation and virustiter in lung tissue at the 6^(th) day, and acted for a longer period oftime than oseltamivir. This compound could still prevent the reductionin arterial oxygen saturation at a dose as low as 1 mg/kg/day. RWJ270201(1 mg/kg/day) could also reduce the death rate, inhibit lungconsolidation and prevent the reduction in arterial oxygen saturation oflaboratory animals infected with lethal influenza virus strain B/HongKong/5/72, and was more effective than oseltamivir. In addition,RWJ270201 at a dose of 10 mg/kg/day could also effectively antagonizethe infection with lethal influenza virus strain A/Bayern/07/95 (H1N1),and reduce the death rate of laboratory animals infected with influenzavirus strain A/NWS/33 (H1N1) (Bantia S., Amold C. S., Parker C. D.,Anti-influenza Virus Activity of Peramivir in Mice with SingleIntramuscular Injection, Antiviral Research, 2006, 69(1), 39-45).RWJ270201 had a low toxicity, and was non-toxic to cells at a dose ashigh as 328 μg/ml; no toxic side effect was found when RWJ270201 wasadministered to rats at a dose of 1000 mg/kg/day for 5 days. No acutetoxic reaction was observed when the dose administered to mice and ratswas up to 3000 mg/kg/day (Sidwell R. W., Smee D. F., Huffman J. H., etal., In the fluenza of Virus Strain, Challenge Dose, and Time of TherapyInitiation on the in vivo Influenza Inhibitory Effect of RWJ270201,Antiviral Res., 2001, 51(3), 179-187). RWJ270201 is a new kind ofhigh-efficiency and low-toxicity NA inhibitor.

However, as shown by study, RWJ270201 is easily hygroscopic, and it isdifficult to completely remove water therein after absorbing moisture,thus the anhydrous form of RWJ270201 is difficult to obtain in industry,and the quality thereof is difficult to effectively control. RWJ270201is unstable in aqueous solution, is easy to self-degrade under theinfluence of environmental factors, and is difficult to directly prepareinto an injection for treating severe patients. The object of theinvention is just for searching for(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid derivative which is not easily hygroscopic and is controllable inquality and a method for preparing the same, and simultaneouslydeveloping a corresponding injection for treating severe patients.

SUMMARY OF THE INVENTION

The present inventors discovered by study that(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate (I) exhibits a good stability, and is not easilyhygroscopic, easily controllable in quality, and suitable forlarge-scale industrial production, as compared to RWJ270201 and otherderivatives.

The results of pharmacodynamic study demonstrated that(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate (I) exhibited the same activity to many types of influenzavirus strains in vivo and in vitro as RWJ270201. The results of drugmetabolism demonstrated that(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate (I) exhibited the same metabolic pathway andpharmacokinetic property as RWJ270201. The results of toxicologicalstudy demonstrated that(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate (I) exhibited the same effect in various toxicity indexesas RWJ270201. Thus,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate (I) is equipotent to RWJ270201.

The present inventors further discovered by study that the stability of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate (I) in aqueous solution can be controlled by adjusting pHvalue of the solution, and a quite good stability can be obtained at asuitable pH value, whereby an injection suitable for treating severepatients is developed.

Therefore, on the one hand, the present invention provides a compound ofthe general formula (I), and a method for preparing it.

On the other hand, the present invention provides a pharmaceuticalcomposition comprising the compound of the invention and a method forpreparing it. The pharmaceutical composition of the invention comprisesat least one compound of the general formula (I), and at least onepharmaceutically acceptable carrier, diluent or excipient.

Still on the other hand, the present invention provides a method fortreating or preventing diseases, risk factors or symptoms caused byinfluenza virus, which comprises administering a therapeutically orpreventively effective amount of the compound of the invention to asubject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate expressed by the general formula (I)

wherein X is 2.0 or 3.0.

The compound of the invention can inhibit the activity of NA ofinfluenza virus.

According to one preferred embodiment, the preferred compound of theinvention is (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate.

According to the invention, the term “influenza virus” includes, but isnot limited to, human influenza viruses and influenza viruses derivedfrom various kinds of animals, e.g., avian influenza virus.

A person skilled in the art shall perceive that the compound of theinvention can also be used in the form of its pharmaceuticallyacceptable salt. The pharmaceutically acceptable salt of the compound ofthe general formula (I) includes conventional salts formed withpharmaceutically acceptable inorganic acids or organic acids orinorganic bases or organic bases and acid addition salts of quaternaryammonium. Examples of suitable salts formed with acids include saltsformed with hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid, perchloric acid, fumaric acid, aceticacid, propionic acid, succinic acid, hydroxyacetic acid, formic acid,lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid,malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid,benzoic acid, salicylic acid, toluenesulfonic acid, methanesulfonicacid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxylnaphthaleneformic acid, hydroiodic acid, malic acid, steroic acid,tannic acid and etc. As for other acids, such as oxalic acid, althoughbeing not pharmaceutically acceptable by themselves, they can be usedfor preparing salts as intermediates, to thereby obtain the compound ofthe invention and pharmaceutically acceptable salts thereof. Examples ofsuitable salts formed with bases include salts formed with sodium,lithium, potassium, magnesium, aluminum, calcium, zinc,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucosamine and procaine. The compound of theinvention as mentioned hereinbelow includes compound of the generalformula (I) and pharmaceutically acceptable salts thereof.

The present invention further includes a prodrug of the compound of theinvention, which, once administered, undergoes a chemical conversionthrough metabolic process, to convert into an active drug. Generally,this kind of drug is a functional derivative of the compound of theinvention, and easily converts into the desired compound of the generalformula (I) in vivo. For example, conventional methods for selecting andpreparing suitable prodrug derivatives are described in “Design OfProdrugs”, H Bund Saard, Elsevier, 1985.

The present invention also includes active metabolites of the compoundof the invention.

On the other hand, the present invention relates to a pharmaceuticalcomposition comprising the compound of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient, which can beused for in vivo treatment and has biocompatibility. The pharmaceuticalcomposition can be prepared into various forms according to differentadministration routes.

The pharmaceutical composition of the invention comprises an effectiveamount of the compound of the general formula (I) or a pharmaceuticallyacceptable salt thereof, and one or more suitable pharmaceuticallyacceptable carrier, diluent or excipient. The pharmaceuticallyacceptable carrier, diluent or excipient includes, but is not limitedto, ion exchanger, aluminum oxide, aluminum stearate, lecithin, serumprotein such as human albumin, buffer substance such as phosphate,glycerin, sorbic acid, potassium sorbate, a mixture of partialglycerides of saturated vegetable fatty acid, water, salt or electrolytesuch as protamine sulfate, disodium hydrogen phosphate, potassiumhydrogen phosphate, sodium chloride, zinc salt, colloidal silica,magnesium trisilicate, polyvinylpyrrolidone, cellulosic material,polyethylene glycol, sodium carboxymethylcellulose, polyacrylate,beeswax, lanolin.

The pharmaceutical composition of the invention may be administered inany of the following routes: orally, spray inhalation, rectally,nasally, buccally, topically, parenterally such as subcutaneous,intravenous, intramuscular, intraperitoneal, intrathecal,intraventricular, intrasternal or encephalic injection or infusion, oradministered with the aid of an explanted reservoir, wherein theadministration routes by parenteral route such as intravenous injectionare preferred.

For oral administration, the compound of the invention may be made intoany orally acceptable preparation forms, including, but not limited to,tablets, capsules, aqueous solutions or aqueous suspensions. Wherein,carrier used in tablets generally includes lactose and corn starch, towhich lubricant such as magnesium stearate may also be added. Diluentused in capsule preparation generally includes lactose and dried cornstarch. Aqueous suspension preparation is generally used by mixingactive ingredient with suitable emulsifying agent and suspending agent.If desired, a sweetening agent, a flavoring agent or a colorant may beadded.

For topical application, in particular for treating the affected face ororgan that is easy to reach by topically external application, e.g.,eyes and skin, the compound of the invention may be made into differentpreparation forms for topical application according to the differentaffected face or organ.

For topical application on eyes, the compound of the invention may bemade into the preparation form of a micronized suspension or solution,wherein the carrier used is an isotonic sterile brine having a certainpH value, and wherein an antiseptic such as chlorinated benzyl alkoxidemay be added or not. For eye use, the compound may also be made into thepreparation form of paste such as vaseline paste.

For topical application on skin, the compound of the invention may bemade into a suitable form of ointment, lotion or cream, wherein theactive ingredient is suspended or dissolved in one or more carrier(s).The carrier used for an ointment includes, but not limited to, mineraloil, liquid vaseline, white vaseline, propylene glycol, polyethyleneoxide, polypropylene oxide, emulsified wax and water; the carrier usedfor a lotion and a cream includes, but not limited to, mineral oil,sorbitan monostearic ester, Tween 60, cetyl esters wax, hexadecylenearomatic alcohol, 2-octyl dodecanol, benzanol and water.

The compound of the invention may also be administered in the form ofsterile injection preparation, including, but not limited to, sterileinjection aqueous or oil suspension or sterile injection solution orsterile powder injection such as lyophilized powder injection. Wherein,the useable carrier and solvent include water, Ringer's solution andisotonic sodium chloride solution. In addition, sterilized non-volatileoil such as monoglyceride or diglyceride may also be used as solvent orsuspending medium. In addition, the preparation may further include a pHadjusting agent, 0.9% aqueous solution of sodium chloride, a buffer, anantioxidant, a metal ion complexing agent, or any combinations thereof.

Further, the administration dosage and manner of the compound of theinvention depend on various factors, such as age, body weight, gender,natural health status and nutrient status of the patient, activity ofthe compound, administration time, metabolic rate, degree of severity ofthe disease, and subjective judgment made by a doctor fordiagnosis/treatment. The administration dosage is preferably within therange of 0.01-100 mg/kg body weight/day, and most preferably within therange of 1-40 mg/kg body weight/day.

The present invention further provides a method for preparing thecompound of the general formula (I).

The patent applications CN1282316A(2001), CN1367776A(2002) disclose amethod for preparing the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product, but the raw materials and the catalysts used thereinare expensive, and hypertoxic benzyl isocyanate is used therein, thusthe method is not suitable for industrial production.

In the present invention, levo-2-azabicyclo[2.2.1]hept-5-en-3-oneprotected with Boc (4) is the starting material, and the intermediate(15) was prepared from the starting material 2-ethyl-butyraldehyde (13)through hydroximation and chlorination. Thereafter, RWJ270201 isprepared by the following seven-step reactions: the intermediate (4) andthe intermediate (15) are subjected to ring closure and hydrolysis toobtain a tert-butylamine salt, which is further subjected to reductionwith NaBH₄/NiCl₂, acetylation with acetic anhydride, removal of Boc withconcentrated hydrochloric acid, hydrolysis with sodium hydroxide, andreaction with 1,2,4-triazole formamidine to get a guanidine substituent.This synthetic route includes 13-step reactions in total. By makingcorresponding improvement based on the original synthetic method, thepresent inventors acquired an improved method for preparing the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product in order to meet the requirement of industrializedproduction.

In the method of the present invention, the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product is dissolved in a solution consisting of water and awater-miscible organic solvent in different ratios under a suitabletemperature typically in the range of 20-100° C., preferably 80-100° C.,wherein the organic solvent includes, but is not limited to, methanol,ethanol, n-butanol, acetone, butanone, tert-butanone and etc., and theratio of water:organic solvent is 100:1-500, or under the condition ofpure water, preferably in the range of 20:1 to 1:2; and then theresulting solution is subjected to crystallization at a suitable coolingrate, e.g., from 0.1° C./min to 5° C./minute, to obtain(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrates (I) containing different crystal water. The compound ofthe general formula (I) of the invention may also be obtained by addingcrystal seed of the compound of the general formula (I) under theaforementioned conditions.

wherein X is 2.0 or 3.0.

The preferred compound of the invention is(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate.

According to the invention, the solubility of the compound of theinvention can be tested by using a standard solubility test method. Forexample, in room-temperature aqueous solution and in neutral buffersolution,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate, as one of the compounds of the general formula (I), hasa solubility 0.17 g/ml, and the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product has a solubility 0.08 g/ml. Therefore, the compoundof the general formula (I) exhibits an obviously increased solubility inaqueous solution and in neutral physiological buffer solution comparedto the corresponding non-hydrate.

The hygroscopicity of the compound of the invention can be tested by astandard hygroscopicity test method. For example, the compound is storedin a sealed vessel at room temperature at a relative humidity of 92.5%,80% or 75%, and the change in moisture content of the compound is testedby sampling at regular intervals. In a ten-day observation test, it isfound that(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate, as one of the compounds of the general formula (I), hasa weight gain of about 2.24%, while the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product has a weight gain of about 13.53%. Therefore, thecompound of the general formula (I) exhibits an obviously reducedhygroscopicity under high wet conditions compared to the correspondingnon-hydrate.

The stability of the compound of the invention can be tested by astandard stability test method, such as the methods specified inGuidelines for Stability Testing of Drug Substances involved in AppendixXIX C of Chinese Pharmacopoeia (2005 edition). For example, thestability of the compound of the general formula (I) can be tested by anaccelerated stability test method, e.g., the stability of the compoundof the general formula (I), at 0, 1, 2, 3, 6 months, is testedrespectively under the conditions: 40° C., 75% relative humidity; 40°C., 92.5% relative humidity; and 80° C. The change in principal agentand relevant substances can be analyzed by thin layer chromatography,high performance liquid chromatography, and etc. In the stability testof 1, 2, 3, 6 months,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate, as one of the compounds of the general formula (I), hasa stable content of the principal agent, and no notable increase in thecontent of the relevant substances, and exhibits a good stability.

The(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrates as obtained in the present invention exhibit a goodstability, and are not easily hygroscopic, and are easily controllablein quality and suitable for large-scale industrialized production.

The present invention further relates to a pharmaceutical compositionfor preventing and treating influenza comprising the compound of thegeneral formula (I) and a pharmaceutically acceptable carrier.

The compound of the general formula (I) herein may be used alone or inthe form of a pharmaceutical composition. The pharmaceutical compositionof the invention can be administered by oral, parenteral or topicaladministration routes. Examples of the administration dosage forminclude, but are not limited to, tablets, capsules, solutions,injections, suppositories, patches, ointments, and etc. The preferreddosage form is the pharmaceutical composition for parenteraladministration and powder injection preparation.

In order to enable the compound of the general formula (I) to besuitable for emergency aid and treatment of severe patients withinfluenza, the present inventors by study developed pharmaceuticalcomposition for parenteral administration and powder injectionpreparation having multiple advantages. Moreover, their preparationmethod is simple, easy to operate, and low in cost.

In the present invention, the amount of the compound of the generalformula (I) or the amount of the compound of the general formula (I) inthe pharmaceutical composition is calculated by(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid.

In concrete, the pharmaceutical composition for parenteraladministration is characterized by comprising a pharmaceuticallyacceptable solvent and from about 50% to about 4000% w/v of the compoundof the general formula (I), wherein the amount of the compound of thegeneral formula (I) in the pharmaceutical composition is calculated bythe amount of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid, and w/v refers to the ratio of the weight of the compound of thegeneral formula (I) to the volume of the solvent in unit dosage form.The (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I) is present in an amount ofpreferably from 300% to 2000% w/v on the basis of the composition. Thecomposition may further optionally comprise at least one ingredientselected from a pH adjusting agent, a buffer, an antioxidant, a metalion complexing agent, or any combinations thereof. The pH adjustingagent therein is selected from inorganic acids, and is present in anamount sufficient to adjust the pH value of the composition to about3-7. The pH adjusting agent is preferably diluted hydrochloric acid, andis preferably present in an amount sufficient to adjust the pH value ofthe composition to about 4-6. The pharmaceutically acceptable solvent isselected from the group consisting of water, 0.9% aqueous solution ofsodium chloride, PEG400, propylene glycol, ethanol, glycerin and anycombinations thereof, preferably water. In the composition whichcomprises water as solvent, the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I) is present in an amount ofpreferably from 300 mg/100 ml to 100 mg/5 ml on the basis of thecomposition.

The powder injection preparation is characterized by comprising the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I), or additionally comprising atleast one of the following pharmaceutically acceptable carriers: a pHadjusting agent, a buffer, an antioxidant, a metal ion complexing agent,or any combinations thereof. The carrier includes, but is not limitedto, dextro-glucoside, mannitol, sodium chloride, sorbitol, citric acidand etc. The commonly used pH adjusting agent includes, but is notlimited to, hydrochloric acid, phosphoric acid, fumaric acid, aceticacid, propionic acid, succinic acid, maleic acid, tartaric acid, citricacid, hydroxymaleic acid, glutamic acid, salicylic acid and etc.,preferably hydrochloric acid. The weight ratio of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate to the pharmaceutically acceptable carrier in unit doseis 1:0-1:100, and the pH adjusting agent is used in an amount sufficientto assure that, when the preparation is used in unit dose, the solutionhas a pH ranging from 2.5 to 6.5, preferably ranging from 3.5 to 5.5.

The above preparation is preferably used for clinical application in theform of intravenous or intramuscular injection, or intravenous dripinfusion.

On the other hand, the present invention further provides a method forpreparing the pharmaceutical composition for parenteral administrationcomprising the (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate, which comprises dissolving the compound of the generalformula (I), adsorbing with activated carbon and filtering, adjustingthe acidity, fine filtrating, filling and high-pressure high-temperaturesterilizing, and etc., or making the compound of the general formula(I), according to a method known in the art, into a powder injectionsuch as lyophilized powder injection.

According to one embodiment in the present invention, the pharmaceuticalcomposition for parenteral administration, per ml, comprises 0.5 mg to 4g of (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I), and at least one of thefollowing pharmaceutically acceptable carriers: a pH adjusting agent,0.9% aqueous solution of sodium chloride, a buffer, an antioxidant, ametal ion complexing agent, or any combinations thereof.

The pharmaceutical composition for parenteral administration comprisingthe(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I) herein may be prepared by thefollowing method comprising the steps of:

(1) dissolving the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I) in a pharmaceutically acceptablesolvent selected from water, 0.9% aqueous solution of sodium chloride,PEG400, propylene glycol, ethanol, glycerin or any combinations thereof,by stirring at room temperature, ultrasonic dissolution, or heating to40-100° C.;

(2) adding 0.01% to 5% of activated carbon at 40-100° C. to carry outadsorption treatment for 10 to 30 minutes, and filtering;

(3) adding a pH adjusting agent in an amount sufficient to adjust the pHvalue of the solution to about 4-6;

(4) filtering with a fine filtration membrane and filling by a volume of1 to 150 ml per unit; and

(5) sealing and sterilizing at 105 to 125° C. for 10 to 50 minutes.

According to another embodiment in the present invention, thepharmaceutical composition for parenteral administration comprising the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I) comprises:

(1) from 300% to 2000% w/v of the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I);

(2) a pharmaceutically acceptable solvent selected from water, PEG400,propylene glycol, ethanol, glycerin or any combinations thereof,preferably water; and

(3) a pH adjusting agent, preferably diluted hydrochloric acid,preferably present in an amount sufficient to adjust the pH value of thecomposition to about 4-6.

According to still another embodiment in the present invention, thepharmaceutical composition for parenteral administration comprising the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I) comprises:

(1) from 300% to 2000% w/v of the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate of the general formula (I);

(2) a pharmaceutically acceptable solvent selected from water, 0.9%aqueous solution of sodium chloride, PEG400, propylene glycol, ethanol,glycerin or any combinations thereof, preferably 0.9% aqueous solutionof sodium chloride; and

(3) a pH adjusting agent, preferably diluted hydrochloric acid,preferably present in an amount sufficient to adjust the pH value of thecomposition to about 4-6.

The most preferred formulation of the pharmaceutical composition forparenteral administration is shown in the examples.

Still on the other hand, the present invention provides a method forpreparing a powder injection preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate, i.e., a powder injection preparation comprising the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate as active ingredient. This powder injection preparationis prepared by dissolving(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate in at least one solvent selected from water forinjection, ethanol and ethyl ether, and then subjecting the resultingsolution to dehydration or desolvation.

According to one embodiment in the present invention, the powderinjection preparation, per gram, comprises from 0.1 mg to 1 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate, or additionally comprises at least one of the followingfilling agents: a pH adjusting agent, a buffer, an antioxidant, a metalion complexing agent, or any combinations thereof.

The powder injection preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate herein may be prepared by the following methodcomprising the steps of:

dissolving(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate with at least one pharmaceutically acceptable solventincluding, but not limited to, water for injection, ethanol and ethylether to obtain a solution having a concentration range of 1-6 g/100 ml,preferably 2.3-3.5 g/100 ml; adjusting the solution with an organic acidor inorganic acid to a pH range of 2.5-6.5, preferably a pH range of3.5-5.5, wherein the inorganic acid and organic acid as commonly usedinclude hydrochloric acid, phosphoric acid, fumaric acid, acetic acid,propionic acid, succinic acid, maleic acid, tartaric acid, citric acid,hydroxymaleic acid, glutamic acid, salicylic acid and etc., preferablyhydrochloric acid; decolorating the solution with a pharmaceuticallyacceptable activated carbon; filtering with a 0.22 μm microporousfiltration membrane; and then drying to obtain a sterile powderinjection preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate, wherein the drying is carried out by a vacuum dryingmethod, a lyophilization method, a spray drying method or a freezing andfilling method.

The pharmaceutical composition for parenteral administration is featuredwith high temperature resistance, storability, high stability, andability of effectively overcoming self-degradation in aqueous solution.The powder injection preparation is featured with advanced preparationmethod, good stability, long period of validity, convenience totransport, and easy to store and preserve. These products have theadvantageous including safe and convenient to use, reliable quality, andeasy to use for emergency aid and treatment of severe patients.

The inhibitory activity of the compound of the general formula (I)against neuraminidase can be assayed according to the method given byBarnett J M and et al (Barnett J M, et al. Antimicrob Agents Chem.,2000; 41, 78-87.). The method is described as follows:

Using 4-MUNANA as a substrate, the activity of influenza virusneuraminidase and the inhibitory activity of the candidate compoundagainst neuraminidase are assayed by fluorimetry. A stock solution offirst generation viruses is cultivated in MDCK cells, and diluted with aneuraminidase assay buffer (NA assay buffer: 32.5 MES, 4 mM CaCl₂, Ph6.5) in a ratio of 1:2. 50 μl of the resulting virus diluted solution ismixed with equal volume of 4-MUNANA (200 mM in NA assay buffer) in ablack 96-well plate (Costar), and incubated at 37° C. for 1 hour, andthen 2 times by volume of a stop buffer (25% ethanol, 0.1M glycine,pH10.7) is added to stop the reaction. The fluorescence intensities at XExcitation: 360 nM and X Emission: 460 nM are assayed (PolarSTAR Optima,BMG Labtech, Germany). A scatter diagram of net value of fluorescenceunit vs. virus concentration is plotted, and different virusconcentrations, generally not less than two, with activity lying in themiddle of the linear part of the scatter diagram are selected and usedfor the assay of the inhibitory activity of the compound. Solutions ofcandidate compounds having different concentrations are formulated usingsterile deionized water. 25 μA of the respective solutions are mixedwith equal amount of viruses (in a concentration two times of thatselected in the activity assay) that have been diluted with 2×NA assaybuffer, followed by acting at room temperature for 30 minutes, and then50 μl of 4-MUNANA (200 mM in NA assay buffer) is added, followed byincubating at 37° C. for 1 hour, and finally the fluorescence intensityis assayed as above described. Each of the concentrations of thecandidate compound is provided in two parallel holes, the blank controlis provided in 4 parallel holes, which only includes 4-MUNANA and thestop buffer, and the virus control is provided in 4 parallel holes,which contains the candidate compound in a concentration of zero. Theinhibitory rate IR (%) is calculated according to the followingequation:

IR (%)=[1−(FU−FUB)÷(FUC−FUB)]×100

FU: average value of fluorescence unit in the candidate compound group;

FUB: average value of fluorescence unit in the blank control group;

FUC: average value of fluorescence unit in the virus control group.

A scatter diagram of IR (%) vs. concentration of compound is plotted,and IC₅₀ is calculated by logarithmic regression analysis. Theregression curves obtained from the assay results of different virusconcentrations shall preferably overlap, with similar IC₅₀ values.

The anti-influenza virus activity of the compound of the general formula(I) can be assayed according to the following method.

A virus drip is prepared typically by including 500 pfu (plaque formingunit) of mice-compliance influenza virus strain in 50 μl phosphatebuffer (containing 0.42% bovine serum albumin), and then dropped intonasal cavity of mice to infect them (BALB/C, female, aged 5-6 weeks,weighted 20 g). The compound of the invention is suspended inphysiological saline to obtain different doses, typically from 2.5 mg/kgto 40 mg/kg, and is administered to the mice at different time beforeand after the viral infection, typically administered by intraperitonealinjection for 5 times, i.e., 1 hour, and 2, 3, 4, 5 days after the viralinfection. The assay is performed by using 8 or 12, typically 10 mice.The result is expressed by the ratio of the number of survival mice the14^(th) day after the viral infection to the number of the mice inassay. Meanwhile, anhydrous (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid is used as a control compound. The results show that the compoundof the invention and the control compound exhibit the same or similaractivity with respect to multiple types of influenza virus strains.

MODE OF CARRYING OUT THE PRESENT INVENTION Examples Example 1 Synthesisof 2-ethyl-N-hydroxybutyrimidoyl chloride (intermediate 15)

1150 g of hydroxylamine hydrochloride, 1000 g of water and 3500 g oftoluene were mixed, to which 1580 g of 2-ethyl-butyraldehyde was addedwith stirring. At 8-12° C., a solution of sodium hydroxide (ca. 30%,4630 g) was added within 1 hour. After completion of the addition, thereaction mixture was further stirred for 60 minutes. Then, the reactionmixture was stratified by standing, and the upper toluene layercontaining compound 14 was taken out and directly used in the followingstep. 4212 g of N-Chlorosuccinimide (NCS) was suspended in 5000 ml ofdimethylformamide, and cooled down to 8° C. The suspension of NCS indimethylformamide was added dropwise to the above toluene solutioncontaining compound 14 within 2.5 hours, whilst keeping the reactiontemperature not exceeding 23° C. After completion of the addition, thereaction mixture was further stirred for 1 hour. Then, 10000 ml of waterwas added, followed by stirring for 30 minutes. The aqueous layer wasdiscarded, and the organic layer was washed with 10000 ml×3 of water.The toluene layer containing compound 15 (chloro-oxime) was separated,and directly used in the following step.

Example 2 Synthesis of tert-butyl ammonium(3aR,4R,6S,6a5)-4-[[(1,1-dimethylethoxy)carbonyl]amino]-3-(1′-ethylpropyl)-3a,5,6,6a-tetrahydro-4H-cyclopenta[d]isooxazole-6-carboxylate(intermediate compound 16)

Methyl(1S,4R)-(−)-methyl-[[(1,1-dimethylethoxy)carbonyl]amino]cyclopent-2-ene-1-carboxylate(4) (2533.2 g), toluene (6000 ml) and triethylamine (4427 ml) were mixedand heated to 50° C. A solution of 2-ethyl-N-hydroxybutyrimidoylchloride (compound 15, 4690 g) in toluene (6000 ml) was added dropwiseto the resulting solution within 2.5 hours. After completion of theaddition, the reaction mixture was further stirred at 62-66° C. forabout 8 hours. 5000 ml of water was added into the reaction solution todissolve solid. The toluene layer was separated, washed with 5000 ml ofwater, and then concentrated to remove about one half of toluene. Asolution of sodium hydroxide (640 g, 16.0 mol) in water (4180 g) wasadded to the resulting concentrated solution, followed by stirring atroom temperature for 7 hours. The toluene layer was discarded, and theaqueous layer was washed with 5000 ml×2 of toluene. 8000 ml of toluenewas added to the aqueous phase. 3710 ml of water was added to 1747 ml ofhydrochloric acid, and the diluted hydrochloric acid was added to theabove toluene layer to neutralize it to a pH of about 4. Then, toluene(about 12000 ml) was added to dissolve solid, and toluene layer wasseparated. At 20-40° C., tert-butylamine (894.0 g) was added dropwise tothe toluene layer, so that a white solid product was precipitated,followed by heating at 95-100° C. for 3 hours, and cooling down to20-25° C. After suction filtration, the resulting solid product waswashed with 8000 ml×2 of acetone, dried at 60° C. for 6 hours. Finally,2694 g of a solid product was obtained, with a yield of 62.0%.

Example 3 Synthesis of methyl(3aR,4R,6S,6aS)-4-[[(1,1-dimethylethoxy)carbonyl]amino]-3-(1′-ethylpropyl)-3a,5,6,6a-tetrahydro-4H-cyclopenta[d]isooxazole-6-carboxylate(5)

1,1-dimethylethyl ammonium(3aR,4R,6S,6aS)-4-[[(1,1-dimethylethoxy)carbonyl]amino]-3-(1′-ethylpropyl)-3a,5,6,6a-tetrahydro-4H-cyclopenta[d]isooxazole-6-carboxylate(16) (3210 g, 7.8 mol) was suspended in acetone (4000 g). Potassiumcarbonate (53.8 g), and an aqueous solution of 30% sodium hydroxidecontaining 312 g of sodium hydroxide were added to the resultingsuspension. 3000 ml of solvent was removed by evaporation, 3000 ml ofacetone was added, then, 3000 ml of solvent was removed by evaporation,3000 ml of acetone was added, and thereafter 4000 ml acetone was removedby evaporation, obtaining a reaction solution substantially free of theodor of tert-butylamine. The reaction solution was cooled down to 30-35°C., to which 1060 ml of dimethyl sulfate was added dropwise at a speedto keep the temperature not exceeding 45° C. After completion of theaddition, the resulting reaction mixture was stirred at 40-45° C. for1.5 hours. The reaction mixture was cooled down to 15-20° C., to whichwas then added 1500 ml of 25% ammonia. After stirring for 30 minutes,1500 ml of methanol was added, and then the reaction mixture was cooleddown to 0-5° C. Thereafter, 2240 ml of 25% ammonia was added to thereaction mixture within 45 minutes. The resulting product was collectedby filtration, washed with 3000 ml of water, and dried under vacuum at45-50° C. Finally, 2686.2 g of a product was obtained, with a yield of97.13%.

Example 4 Synthesis of methyl (1S,2S,3S,4R,1′S)-3-[(1-amino-2′-ethyl)butyl]-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxycyclopentane-1-carboxylate(6)

Intermediate 5 (1000 g) and nickel chloride hexahydrate (700 g) weredissolved in methanol (2500 ml), and cooled down to −10˜5° C. Sodiumhydroxide (5 g) and sodium borohydride (300 g) were dissolved inmethanol (2300 ml), and added to the above reaction solution withinabout 4-6 hours, whilst keeping the temperature of the reaction solutionin the range of −10˜4° C. After completion of the addition, the reactionsolution was stirred at 0˜5° C. for 60 minutes. A solution formed bysodium nitrite (200 g), ammonium chloride (560 g) and 25% ammonia (650g) with water (6000 ml) was added to the reaction solution. The reactionsolution was then stirred at room temperature for 16 hours. Thereafter,the reaction solution was subjected to suction filtration, and thefilter cake obtained was washed with a solution formulated by 25%ammonia (340 g) and water (2500 g) for 2 times. The filter cake wassuspended in toluene (15000 ml) and 25% ammonia (1250 g), and stirred at75˜80° C. for 60 minutes. The organic phase was separated, to which wasadded 25% ammonia (1500 g). Then, a solution of disodiumethylenediaminetetraacetate dihydrate (EDTA) (150 g) in water (2000 g)was added. After heating at 70˜80° C. for 60 minutes, the organic layerwas separated. The organic layer was cooled down to 0˜5° C., stirred for2-3 hours, and vacuum filtered. The filter cake obtained was washed withtoluene (2000 g), and dried under vacuum at 40˜50° C., obtaining a whitesolid compound 6, 740 g (73.2%).

Example 5 Synthesis of the (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product (9)

Procedures

2000 g of intermediate 6 was suspended in 10000 ml of toluene, andcooled down to 2˜5° C., to which was added dropwise 680 g of aceticanhydride. After completion of the addition, the reaction mixture wasstirred at room temperature for 1.5 hours. Then, 4800 ml of 10% aqueoussolution of sodium carbonate was slowly added, followed by stirring for15 minutes. The aqueous layer was discarded, and the organic layer,i.e., a toluene solution containing intermediate 7, was directly used inthe subsequent step.

In an ice bath, 1900 ml of concentrated hydrochloric acid was addeddropwise to the above toluene solution. After completion of theaddition, the reaction mixture was stirred for 1.5 hours. Afterstratification, the organic layer was washed with water (1000 ml) forone time. The aqueous layers were combined, obtaining an aqueoussolution containing intermediate 8, which was directly used in thesubsequent step.

At 0˜6° C., 2920 ml of 30% aqueous solution of sodium hydroxide wasadded to the above aqueous solution. After completion of the addition,the solution was further stirred for 60 minutes, obtaining an aqueoussolution containing intermediate 21, which was directly used in thesubsequent step.

1020 g of 1,2,4-triazole-1-formamidine hydrochloride was added to theabove aqueous solution containing intermediate 21, followed by stirringfor 60 minutes. Then, 30% aqueous solution of sodium hydroxide was addedto adjust the pH of the reaction solution to 8.4. The reaction solutionwas stirred at room temperature overnight, and then stirred at 0˜4° C.for 4 hours. Finally, the reaction solution was subjected to suctionfiltration and then dried, obtaining 1150 g of a white solid, the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product, with a yield of 30%.

Example 6 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid monohydrate

1200 g of the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product was suspended in 20000 ml of water, and dissolved byheating to 90° C. After standing for a moment, 50 g of activated carbonwas added to the system, followed by refluxing at 90° C. for 10 minutes,and filtering while the system was hot. After cooling down by standingto 75° C., 6000 ml of acetone was added to the filtrate. In an ice bath,the mixed solution was rapidly cooled down with stirring, so that alarge amount of white powdery solid was precipitated. After cooling downto about 4° C., the mixed system was moved to a refrigerator for coldstorage. After 12 hours, the mixed system was filtered, and the filtercake obtained was washed with a mixture of acetone and water. Theobtained solid was naturally dried, obtaining 1100 g of a white solidproduct, (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid monohydrate, with a purity of 99.57% and a moisture content of5.31%.

Example 7 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid dihydrate

1000 g of the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product was suspended in 30000 ml of water, and dissolved byheating to 90° C. After standing for a moment, 50 g of activated carbonwas added to the system, followed by refluxing at 90° C. for 10 minutes,and filtering while the system was hot. After cooling down by standingto 75° C., 6000 ml of methanol was added to the filtrate. Then, themixed solution was placed in a desiccator and sealed, and then placed ina temperature-controlled reaction tank wherein the temperature wascontrolled to decrease from 75° C. to −10° C. within 6 hours, so that alarge amount of white powdery solid was continuously precipitated. Afterisothermally standing for 12 hours, the mixed system was filtered, andthe filter cake obtained was washed with a mixture of methanol andwater. The obtained solid was naturally dried, obtaining 880 g of awhite solid product,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid dihydrate, with a purity of 99.67% and a moisture content of 9.95%.

Example 8 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate

1200 g of the (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product was suspended in 30000 ml of water, and dissolved byheating to 90° C. After standing for a moment, 50 g of activated carbonwas added to the system, followed by refluxing at 90° C. for 10 minutes,and filtering while the system was hot. After cooling down by standingto 75° C., 5000 ml of methanol was added to the filtrate. Then, themixed solution was placed in a desiccator and sealed. After standing forseveral hours, a large amount of white powdery solid was precipitated.After 12 hours, the mixed system was filtered, and the filter cakeobtained was washed with a mixture of methanol and water. The obtainedsolid was naturally dried, obtaining 1080 g of a white solid product,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate, with a purity of 99.86% and a moisture content of14.21%.

Example 9 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid semihydrate

100 g of the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product was suspended in 1000 ml of methanol, and dissolvedby heating to 70° C. After standing for a moment, 5 g of activatedcarbon was added to the system, followed by refluxing at 70° C. for 10minutes, and filtering while the system was hot. 1000 ml of water wasadded to the filtrate. In an ice bath, the mixed solution was rapidlycooled down with stirring, so that a large amount of white powdery solidwas precipitated. After cooling down to about 4° C., the mixed systemwas moved to a refrigerator for cold storage. After 12 hours, the mixedsystem was filtered, and the filter cake obtained was washed with amixture of methanol and water. The obtained solid was naturally dried,obtaining 84 g of a white solid product,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid semihydrate, with a purity of 99.71% and a moisture content of2.73%.

Example 10 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid sesquihydrate

100 g of the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product was suspended in 1000 ml of acetone, and dissolved byheating to 70° C. After standing for a moment, 5 g of activated carbonwas added to the system, followed by refluxing at 70° C. for 10 minutes,and filtering while the system was hot. 1200 ml of water was added tothe filtrate. Then, the temperature of the mixed solution was controlledto decrease from 75° C. to 4° C. within 5 hours, so that a large amountof white powdery solid was precipitated. After cooling down to about 4°C., the mixed system was moved to a refrigerator for cold storage. After12 hours, the mixed system was filtered, and the filter cake obtainedwas washed with a mixture of acetone and water. The obtained solid wasnaturally dried, obtaining 80 g of a white solid product,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid sesquihydrate, with a purity of 99.61% and a moisture content of7.64%.

Example 11 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hexahydrate

100 g of the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid crude product was suspended in 1300 ml of water, and dissolved byheating to 100° C. After standing for a moment, 5 g of activated carbonwas added to the system, followed by refluxing at 100° C. for 10minutes, and filtering while the system was hot. The temperature of thefiltrate was controlled to decrease from 75° C. to 5° C. within 1 hour,so that a large amount of white powdery solid was precipitated. Aftercooling down to about 4° C., the mixed system was moved to arefrigerator for cold storage. After 12 hours, the mixed system wasfiltered, and the filter cake obtained was washed with a mixture ofmethanol and water. The obtained solid was naturally dried, obtaining 78g of a white solid product,(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hexahydrate, with a purity of 99.59% and a moisture content of24.92%.

Example 12 Determination of solubility of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates

According to Chinese Pharmacopoeia (2005 edition), Vol. II, GeneralNotice 5(2), (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates, which had been grounded into fine powder, wereweighed in a suitable amount, and put into different volumes of solventat 25±2° C. While vigorously stirring for 30 seconds every 5 minutes,the dissolution of the powder was observed within 30 minutes. When nopowder particle of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates was observed, it was considered that the powderwas completely dissolved. The results were shown in the following table.

The determination results of solubility of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates

Solvent Samples Solubility (g/ml) Water Anhydrous 0.08 Monohydrate 0.15Sesquihydrate 0.03 Dihydrate 0.05 Trihydrate 0.17 Hexahydrate 0.11

Example 13 Determination of hygroscopicity of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates

According to the method for investigating influencing factors of highhumidity as specified in Guidelines for Stability Testing of DrugSubstances involved in Appendix XIX C of Chinese Pharmacopoeia (2005edition),(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates were placed in a humidity-constant sealed vessel,under the conditions of 25° C. and a relative humidity of 90%±5%, for 10days. At the 5^(th) and 10^(th) days, the products were sampled anddetermined in aspect of important items relating to stabilityinvestigation. Meanwhile, the weights of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates, before and after the experiment, were exactlyweighed, to thereby investigate the hygroscopicity and deliquescence ofthe products. If the weight gain of a product due to moisture absorptionwas more than 5%, the product was further experimented according to thesame method under a relative humidity of 75%±5%. If the weight gain of aproduct due to moisture absorption was less than 5%, and other itemsthereof under investigation met the relevant requirements, no moreexperiment was needed. The humidity-constant condition could be achievedby placing a saturated saline solution at the lower part of a sealedvessel, e.g., a desiccator, and a NaCl saturated solution (15.5-60° C.,a relative humidity of 75%±1%) or a KNO₃ saturated solution (25° C., arelative humidity of 92.5%) may be selected according to the requirementof different relative humidity. The results were showed in the followingtable.

The determination results of hygroscopicity of (1S,2S,3S,4R)-3-[(1S)-1-cetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid and its hydrates

Hygro- Hygro- scopicity scopicity Relative at the 5^(th) at the 10^(th)humidity Samples day (%) day (%) Remarks 92.5% Anhydrous 8.38 13.53 Notconform to the requirement Monohydrate 2.83 3.35 Not conform to therequirement Sesquihydrate 4.48 7.33 Not conform to the requirementDehydrate 2.54 2.98 Conform to the requirement Trihydrate 2.11 2.24Conform to the requirement Hexahydrate 4.37 9.96 Not conform to therequirement  75% Anhydrous 7.23 12.88 Not conform to the requirementMonohydrate 2.41 2.77 Conform to the requirement Sesquihydrate 4.01 6.89Not conform to the requirement Dehydrate 2.21 2.54 Conform to therequirement Trihydrate 1.68 1.83 Conform to the requirement Hexahydrate3.86 6.33 Not conform to the requirement

Example 14 Investigation of stability of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrates

According to the accelerated test method as specified in Guidelines forStability Testing of Drug Substances involved in Appendix XIX C ofChinese Pharmacopoeia (2005 edition), 3 batches of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrates, in packages for sale, were placed under the conditions ofa temperature of 40° C.±2° C. and a relative humidity of 75%±5% for 6months. The device used should be capable of controlling temperaturewith a precise of ±2° C., and relative humidity with a precise of ±5%,and could monitor true temperature and humidity. The products weresampled respectively at the end of the 1, 2, 3 and 6 months during thetest, and determined in aspect of important items relating to stabilityinvestigation. If the sample did not meet the specified quality standardas determined within 6 months under the above conditions, it should besubjected to accelerated experiment under intermediate conditions, i.e.,a temperature of 30° C.±2° C. and a relative humidity of 60%±5% (NaNO₂saturated solution (25-40° C., a relative humidity of 64-61.5%) might beused), for 6 months.

The investigation results of stability of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrates

Investigation items Change in Relevant Com- Time weight Contentsubstances pound (month) Character (%) (%) (%) Trihydrate 0 White solid— 99.90 Conform to the powder requirement 1 White solid +0.01 99.95Conform to the powder requirement 2 White solid −0.02 99.97 Conform tothe powder requirement 3 White solid +0.01 99.96 Conform to the powderrequirement 6 White solid +0.01 99.91 Conform to the powder requirement

Example 15 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate injection with specification of 100 mg

Components Weight (g)(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 116.4guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate Water forinjection 4868.6 Activated carbon 5 Diluted hydrochloric acid (0.1M) 10Total amount 5000

116.4 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate was dissolved in 4000 ml water for injection at 80° C.till complete dissolution. Thereafter, 5 g of activated carbon was addedto the resulting solution while it was hot, to effect adsorption at 80°C. for 20 minutes. The solution was filtered, and the resulting filtratewas supplemented with water for injection to a total volume of 5000 ml.After the filtrate was cooled down to room temperature, 10 ml of 0.1 Mdiluted hydrochloric acid was added to adjust its pH to 3-5, followed byfiltration using a filtration membrane of 0.2 μm. The resulting filtratewas filled into ampoules (1000 in total) in an amount of 5 ml perampoule. After completion of the filling, the ampoules were sealed, andsterilized at 115° C. for 20 minutes.

Example 16 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate injection with specification of 50 mg

Components Weight (g)(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 58.2guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate Water forinjection 4926.8 Activated carbon 5 Diluted hydrochloric acid (0.1M) 10Total amount 5000

58.2 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate was dissolved in 4000 ml water for injection at 80° C.till complete dissolution. Thereafter, 5 g of activated carbon was addedto the resulting solution while it was hot, to effect adsorption at 80°C. for 20 minutes. The solution was filtered, and the resulting filtratewas supplemented with water for injection to a total volume of 5000 ml.After the filtrate was cooled down to room temperature, 10 ml of 0.1 Mdiluted hydrochloric acid was added to adjust its pH to 3-5, followed byfiltration using a filtration membrane of 0.2 μm. The resulting filtratewas filled into ampoules (1000 in total) in an amount of 5 ml perampoule. After completion of the filling, the ampoules were sealed, andsterilized at 115° C. for 20 minutes.

Example 17 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate injection with specification of 300 mg

Components Weight (g)(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 34.9guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate Water forinjection 9850.1 Sodium chloride 90 Activated carbon 10 Sodium bisulfite10 Sodium calciumedetate 5 Total amount 10000

90 g of sodium chloride, 10 g of sodium bisulfite and 5 g of sodiumcalciumedetate were dissolved in 8000 ml water for injection at 80° C.till complete dissolution. Then, 34.9 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate was further added therein till complete dissolution.Thereafter, 10 g of activated carbon was added to the resulting solutionwhile it was hot, to effect adsorption at 80° C. for 20 minutes. Thesolution was filtered, and the resulting filtrate was supplemented withwater for injection to a total volume of 10000 ml. After cooling down toroom temperature, the filtrate was further filtered using a filtrationmembrane of 0.2 μm. The resulting filtrate was filled into ampoules (100in total) in an amount of 100 ml per ampoule. After completion of thefilling, the ampoules were sealed, and sterilized at 115° C. for 20minutes.

Example 18 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate injection with specification of 300 mg

Components Weight (g)(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 349guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate Water forinjection 98366 Sodium chloride 900 Activated carbon 100 Dilutedhydrochloric acid (1M) 285 Total amount 100000

900 g of sodium chloride was dissolved in 80000 ml water for injectionat 80° C. till complete dissolution. Then, 349 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate was further added therein till complete dissolution.Thereafter, 100 g of activated carbon was added to the resultingsolution while it was hot, to effect adsorption at 80° C. for 20minutes. The solution was filtered to remove the activated carbon, and285 ml of 1M diluted hydrochloric acid was added to the resultingfiltrate to adjust its pH to 4-6. Then, the filtrate was supplementedwith water for injection to a total volume of 100000 ml. After coolingdown to room temperature, the filtrate was further filtered using afiltration membrane of 0.2 μm. The resulting filtrate was filled intoampoules (1000 in total) in an amount of 100 ml per ampoule. Aftercompletion of the filling, the ampoules were sealed, and sterilized at115° C. for 20 minutes.

Example 19 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate injection with specification of 500 mg

Components Weight (g)(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 582guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate Water forinjection 98133 Sodium chloride 900 Activated carbon 100 Dilutedhydrochloric acid (1M) 285 Total amount 100000

900 g of sodium chloride was dissolved in 80000 ml water for injectionat 80° C. till complete dissolution. Then, 582 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate was further added therein till complete dissolution.Thereafter, 100 g of activated carbon was added to the resultingsolution while it was hot, to effect adsorption at 80° C. for 20minutes. The solution was filtered to remove the activated carbon, and285 ml of 1M diluted hydrochloric acid was added to the resultingfiltrate to adjust its pH to 4-6. Then, the filtrate was supplementedwith water for injection to a total volume of 100000 ml. After coolingdown to room temperature, the filtrate was further filtered using afiltration membrane of 0.2 μm. The resulting filtrate was filled intoampoules (1000 in total) in an amount of 100 ml per ampoule. Aftercompletion of the filling, the ampoules were sealed, and sterilized at115° C. for 20 minutes.

Example 20 Preparation of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate powder injection preparation

Formula 1

(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 23.3 gguanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate (equivalentto peramivir) 20.0 g Sodium chloride  9.0 g 0.1N diluted hydrochloricacid solution adjusting pH to 3.5-5 Water added till 1000 ml 200 bottleswere prepared with specification of 100 mg principal agent per bottle

23.3 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate and 9 g of sodium chloride were weighed in a clean room,and placed in a sterilized vessel, to which was added 500 ml ofsterilized water for injection. Then, the resulting solution wasadjusted with 0.1 N diluted hydrochloric acid solution to a pH of 3.5-5,and supplemented with sterilized water for injection till 1000 ml, andthen adsorbed with 1 g of activated carbon. After filtration, theresulting filtrate was further filtered with a sterilizer equipped witha 0.22 μm of microfiltration membrane. The solution as finally obtainedwas filled into 20 ml sterilized penicillin bottles in an amount of 5 mlper bottle. The bottles were subjected to pre-freezing at −30° C.˜-40°C. for 2-3 hours, sublimation drying at −36° C.˜-20° C. for 9 hours,drying at 30° C. for 10-24 hours, capping, rolling-installing, andpackaging, whereby the powder injection preparation was prepared.

Formula 2

The formula and the preparation method were the same as those of thegeneral formula 1, except for that the solution as formulated wassubjected to sterile drying in a large dish, followed by crushing, splitcharging in penicillin bottles in specified weight, capping, sealing,and packaging.

Formula 3

(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 23.3 gguanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate (equivalentto peramivir) 20.0 g mannitol 33.6 g 0.1N diluted hydrochloric acidsolution adjusting pH to 3.5-5 Water added till 1000 ml 200 bottles wereprepared with specification of 100 mg principal agent per bottle

23.3 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate and 33.6 g of mannitol were weighed in a clean room, andplaced in a sterilized vessel, to which was added 500 ml of sterilizedwater for injection. Then, the resulting solution was adjusted with 0.1N diluted hydrochloric acid solution to a pH of 3.5-5, and supplementedwith sterilized water for injection till 1000 ml, and then adsorbed with1 g of activated carbon. After filtration, the resulting filtrate wasfurther filtered with a sterilizer equipped with a 0.22 μm ofmicrofiltration membrane. The solution as finally obtained was filledinto 20 ml sterilized penicillin bottles in an amount of 5 ml perbottle. The bottles were subjected to pre-freezing at −30° C.˜-40° C.for 2-3 hours, sublimation drying at −36° C.˜-20° C. for 9 hours, dryingat 30° C. for 10-24 hours, capping, rolling-installing, and packaging,whereby the powder injection preparation was prepared.

Formula 4

The formula and the preparation method were the same as those of thegeneral formula 3, except for that the solution as formulated wassubjected to sterile drying in a large dish, followed by crushing, splitcharging in penicillin bottles in specified weight, capping, sealing,and packaging.

Formula 5

(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 23.3 gguanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate (equivalentto peramivir) 20.0 g Sodium chloride  9.0 g 0.1N diluted acetic acidsolution adjusting pH to 3.5-5 Water added till 1000 ml 200 bottles wereprepared with specification of 100 mg principal agent per bottle

23.3 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate and 9 g of sodium chloride were weighed in a clean room,and placed in a sterilized vessel, to which was added 500 ml ofsterilized water for injection. Then, the resulting solution wasadjusted with 0.1 N diluted acetic acid solution to a pH of 3.5-5, andsupplemented with sterilized water for injection till 1000 ml, and thenadsorbed with 1 g of activated carbon. After filtration, the resultingfiltrate was further filtered with a sterilizer equipped with a 0.22 μmof microfiltration membrane. The solution as finally obtained was filledinto 20 ml sterilized penicillin bottles in an amount of 5 ml perbottle. The bottles were subjected to pre-freezing at −30° C.˜-40° C.for 2-3 hours, sublimation drying at −36° C.˜-20° C. for 9 hours, dryingat 30° C. for 10-24 hours, capping, rolling-installing, and packaging,whereby the powder injection preparation was prepared.

Formula 6

The formula and the preparation method were the same as those of thegeneral formula 5, except for that the solution as formulated wassubjected to sterile drying in a large dish, followed by crushing, splitcharging in penicillin bottles in specified weight, capping, sealing,and packaging.

Formula 7

(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 23.3 gguanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate (equivalentto peramivir) 20.0 g 0.1N diluted hydrochloric acid solution adjustingpH to 3.5-5 Water added till 1000 ml 200 bottles were prepared withspecification of 100 mg principal agent per bottle

23.3 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate was weighed in a clean room, and placed in a sterilizedvessel, to which was added 500 ml of sterilized water for injection.Then, the resulting solution was adjusted with 0.1 N diluted acetic acidsolution to a pH of 3.5-5, and supplemented with sterilized water forinjection till 1000 ml, and then adsorbed with 1 g of activated carbon.After filtration, the resulting filtrate was further filtered with asterilizer equipped with a 0.22 μm of microfiltration membrane. Thesolution as finally obtained was filled into 20 ml sterilized penicillinbottles in an amount of 5 ml per bottle. The bottles were subjected topre-freezing at −30° C.˜-40° C. for 2-3 hours, sublimation drying at−36° C.˜-20° C. for 9 hours, drying at 30° C. for 10-24 hours, capping,rolling-installing, and packaging, whereby the powder injectionpreparation was prepared.

Formula 8

The formula and the preparation method were the same as those of thegeneral formula 7, except for that the solution as formulated wassubjected to sterile drying in a large dish, followed by crushing, splitcharging in penicillin bottles in specified weight, capping, sealing,and packaging.

Formula 9

(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 34.95 gguanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate (equivalentto peramivir) 30.0 g Sodium chloride  9.0 g 0.1N diluted hydrochloricacid solution adjusting pH to 3.5-5 Water added till 1000 ml 200 bottleswere prepared with specification of 150 mg principal agent per bottle

34.95 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate and 9 g of sodium chloride were weighed in a clean room,and placed in a sterilized vessel, to which was added 500 ml ofsterilized water for injection. Then, the resulting solution wasadjusted with 0.1 N diluted hydrochloric acid solution to a pH of 3.5-5,and supplemented with sterilized water for injection till 1000 ml, andthen adsorbed with 1 g of activated carbon. After filtration, theresulting filtrate was further filtered with a sterilizer equipped witha 0.22 μm of microfiltration membrane. The solution as finally obtainedwas filled into 20 ml sterilized penicillin bottles in an amount of 5 mlper bottle. The bottles were subjected to pre-freezing at −30° C.˜-40°C. for 2-3 hours, sublimation drying at −36° C.˜-20° C. for 9 hours,drying at 30° C. for 10-24 hours, capping, rolling-installing, andpackaging, whereby the powder injection preparation was prepared.

Formula 10

The formula and the preparation method were the same as those of thegeneral formula 9, except for that the solution as formulated wassubjected to sterile drying in a large dish, followed by crushing, splitcharging in penicillin bottles in specified weight, capping, sealing,and packaging.

Formula 11

(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4- 34.95 gguanidino-2-hydroxy-cyclopentyl-1-carboxylic acid trihydrate (equivalentto peramivir) 30.0 g Sodium chloride  9.0 g 0.1N diluted hydrochloricacid solution adjusting pH to 3.5-5 Water added till 1000 ml 100 bottleswere prepared with specification of 300 mg principal agent per bottle

34.95 g of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate and 9 g of sodium chloride were weighed in a clean room,and placed in a sterilized vessel, to which was added 500 ml ofsterilized water for injection. Then, the resulting solution wasadjusted with 0.1 N diluted hydrochloric acid solution to a pH of 3.5-5,and supplemented with sterilized water for injection till 1000 ml, andthen adsorbed with 1 g of activated carbon. After filtration, theresulting filtrate was further filtered with a sterilizer equipped witha 0.22 μm of microfiltration membrane. The solution as finally obtainedwas filled into 50 ml sterilized penicillin bottles in an amount of 10ml per bottle. The bottles were subjected to pre-freezing at −30°C.˜-40° C. for 2-3 hours, sublimation drying at −36° C.˜-20° C. for 9hours, drying at 30° C. for 10-24 hours, capping, rolling-installing,and packaging, whereby the powder injection preparation was prepared.

Example 20 Investigation of dissolution behavior of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate in water

A pre-test was carried out with the specification of 100 ml:300 mgwherein the raw material is the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate and the dissolution conditions were as follows:

(1) water for injection (room temperature)

(2) water for injection (80° C.)

(3) water for injection (room temperature, adjusted to be acidic withhydrochloric acid)

(4) water for injection (adjusted to be basic with disodium hydrogenphosphate)

Results:

Under the condition (1), the compound was slowly dissolved while usingwater for injection in its total amount, with stirring or ultrasonicdissolution for a relatively long period of time. Under the condition(2), the compound was rapidly dissolved while using water for injectionin a relatively little volume; considering the fact that water forinjection is generally circulated at 80° C. during scale production,this condition is the preferred condition for the convenience ofproduction. Under the condition (3), the compound was relatively easilydissolved, and as compared with the case under the condition (4), thecompound was more easily dissolved under acidic condition, which couldserve as a basis for the adjustment of pH value.

Example 21 Influence of different acidic conditions on the stability of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate

349 mg of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate and 900 mg of sodium chloride were dissolved with waterfor injection at 80° C. according to the dissolution behavior of thetrihydrate, and adjusted with hydrochloric acid to different pH values.The resulting solutions were high pressure steam sterilized at 115° C.for 30 minutes. Then, the solutions were sampled for the determinationof change in pH value and change in amount of relevant substances. Theresults were listed as follows:

Table of pH determination pH value Amount (relevant substances) No. ofBefore After Before After After 10 days solution Appearancesterilization sterilization sterilization sterilization at 60° C. 1Colorless 4.00 4.08 98.58 (0.11) 97.8 (0.17) 101.20 (0.10)  transparent(2 impure liquid peaks) 2 Colorless 4.53 4.50 102.90 (0.11)  99.3 (0.10) 96.1 (0.09) transparent liquid 3 Colorless 5.02 5.02 101.1 (0.10) 101.7(0.10)  100.4 (0.10) transparent liquid 4 Colorless 5.40 5.40 104.3(0.29) 101.7 (0.10)  102.8 (0.09) transparent (2 impure liquid peaks) 5Colorless 6.08 6.04 103.8 (0.11) 102.5 (0.10)  104.0 (0.10) transparentliquid 6 Colorless 6.56 6.30 104.2 (0.10) −(0.10) 104.4 (0.39)transparent (5 impure liquid peaks) 7 Colorless 6.95 6.40 102.90 (0.10) 95.7 (0.10) 93.43 (0.11) transparent liquid 8 Colorless 7.53 6.72 104.1(0.10) 98.8 (0.09) 103.9 (0.16) transparent (3 impure liquid peaks) 9Colorless 7.93 7.09 104.1 (0.20) 94.3 (0.10) 102.73 (0.27)  transparent(3 impure liquid peaks) Notes: the data inside the parentheses was theamount of relevant substances, and calculated by normalization method.The data outside the parentheses was the amount of the trihydrate.

The results showed that: when the pH value exceeded 6.5, it was reducedafter sterilization, and the extent of reduction was increased as the pHvalue increased, which indicated that the pH value of the system couldbe changed by high temperature when said pH value was high. With respectto the change in amount, when the pH value exceeded 7.0, the amountafter sterilization was obviously reduced, and continuously reducedafter 10 days at 60° C.; when the pH value was 4.5, the amount alsotended to reduce after 10 days at 60° C. This indicated that either toohigh or too low pH value exerted an adverse influence on the stabilityof the system. The analysis on relevant substances indicated that, ascalculated by normalization method, the amount of the relevantsubstances had no obvious change, but the number of impure peaksexhibited that the number of relevant substances tended to increase asthe pH value became more basic. Therefore, the compound tended to bestable under acidic conditions. The optimum pH value was within therange of 4.5-6.5.

Example 22 Study on Conditions of Adsorption and Sterilization withActivated Carbon

The change in amount before and after adsorption with activated carbon,used in amounts of 0.05%, 0.1% and 0.2% respectively, was investigated.The results showed that the adsorption amount was increased as theamount of activated carbon increased, and the adsorption amount wasabout 2% when the amount of activated carbon was 0.1%. In order toassure the quality of the product, activated carbon was used in anamount of 0.1%. Sterilization was performed under two conventionalconditions, i.e., 115° C., 30 minutes and 121° C., 20 minutes,respectively. The results showed that the amount was somewhat reducedunder the condition of 121° C., 20 minutes, and the use of aconventional sterilization method under the condition of 115° C., 30minutes just could result in F0>8.

Example 23 Study on optimizing the formula of pharmaceutical compositionfor parenteral administration comprising the(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate

According to the basic formula:

Trihydrate 349 mg Sodium chloride 0.9 g Water for injection 100 ml

three samples were prepared as follows: by adjusting the pH value withdiluted hydrochloric acid, the sample prepared with pH=5.0 was marked asSY1, the sample prepared with pH=6.0 was marked as SY2, and the sampleprepared with pH=6.5 was marked as SY3. The samples prepared weresubjected to high temperature, illumination experiments, and 40° C.accelerated experiment.

According to the basic formula:

Trihydrate 116.4 mg Water for injection 5 ml

three samples were prepared as follows: by adjusting the pH value withhydrochloric acid to 5.04, the sample prepared was marked as XZ1; byadding 0.05% thiourea and adjusting the pH value with ammonia to 6.98,the sample prepared was marked as XZ2; by directly dissolving trihydratein water for injection without the addition of any adjuvant and pHadjusting agent, the sample prepared was marked as XZ3. The samplesprepared were subjected to high temperature, illumination experiments,and 40° C. accelerated experiment.

The results were showed in the following table:

Before Illumination Illumination 60° C. 60° C. 40° C. Samplesterilization 0 d 5 d 10 d 5 d 10 d 10 d pH value SY1 5.07 5.06 5.085.10 5.08 5.10 5.06 SY2 5.95 5.99 6.03 6.01 6.04 6.07 6.03 SY3 6.20 6.396.21 6.22 6.29 6.31 6.29 XZ1 5.04 5.03 5.05 5.03 5.06 5.05 5.05 XZ2 6.986.88 6.88 6.90 6.91 6.86 6.89 XZ3 7.22 6.98 6.99 6.97 6.96 6.95 6.93Amount (%) SY1 101.2 100.5 99.5 100.2 99.3 100.9 100.2 SY2 100.8 99.699.0 99.5 100.1 99.2 99.1 SY3 102.5 99.8 99.1 98.2 99.1 98.2 99.6 XZ198.5 97.8 98.0 98.2 99.6 100.9 100.2 XZ2 100.0 98.6 99.7 98.5 97.1 98.099.8 XZ3 101.1 97.5 98.1 97.8 98.1 98.3 99.9 Relevant substances (%) SY10.10 0.11 0.09 0.11 0.12 0.09 0.10 SY2 0.09 0.13 0.12 0.18 0.13 0.100.12 SY3 0.13 0.15 0.17 0.20 0.15 0.18 0.11 XZ1 0.13 0.12 0.11 0.10 0.120.16 0.11 XZ2 0.12 0.14 0.15 0.18 0.09 0.14 0.10 XZ3 0.11 0.18 0.20 0.110.19 0.27 0.18

Example 24 Determination of inhibitory activity of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate against influenza A virus neuraminidase

Using 4-MUNANA as a substrate, the activity of influenza virusneuraminidase and the inhibitory activity of the candidate compoundagainst neuraminidase were assayed by fluorimetry. A stock solution offirst generation viruses was cultivated in MDCK cells, and diluted witha neuraminidase assay buffer (NA assay buffer: 32.5 MES, 4 mM CaCl₂,pH6.5) in a ratio of 1:2. 50 μl of the resulting virus diluted solutionwas mixed with equal volume of 4-MUNANA (200 mM in NA assay buffer) in ablack 96-well plate (Costar), and incubated at 37° C. for 1 hour, andthen 2 times by volume of a stop buffer (25% ethanol, 0.1M glycine, pH10.7) was added to stop the reaction. The fluorescence intensities atExcitation: 360 nM and Emission: 460 nM were assayed (PolarSTAR Optima,BMG Labtech, Germany). A scatter diagram of net value of fluorescenceunit vs. virus concentration was plotted, and two virus concentrationswith activity lying in the middle of the linear part of the scatterdiagram were selected and used for the assay of the inhibitory activityof the compound. Solutions of (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate having concentrations of 0.01, 0.1, 1, 10, 100 pMol wererespectively formulated using sterile deionized water. 25 μl of therespective solutions were mixed with equal amount of viruses (in aconcentration two times of that selected in the activity assay) that hadbeen diluted with 2×NA assay buffer, followed by acting at roomtemperature for 30 minutes, and then 50 μl of 4-MUNANA (200 mM in NAassay buffer) was added, followed by incubating at 37° C. for 1 hour,and finally the fluorescence intensity was assayed as above described.Each of the concentrations of the candidate compound was provided in twoparallel holes, the blank control was provided in 4 parallel holes,which only included 4-MUNANA and the stop buffer, and the virus controlwas provided in 4 parallel holes, which contained the candidate compoundin a concentration of zero. The inhibitory rate IR (%) was calculatedaccording to the following equation:

IR (%)=[1−(FU−FUB)÷(FUC−FUB)]×100

FU: average value of fluorescence unit in the candidate compound group;

FUB: average value of fluorescence unit in the blank control group;

FUC: average value of fluorescence unit in the virus control group.

A scatter diagram of IR (%) vs. concentration of compound was plotted,and IC₅₀ was calculated by logarithmic regression analysis. Theregression curves obtained from the assay results of different virusconcentrations should preferably overlap, with similar IC₅₀ values. Theresults were listed as follows:

Inhibitory activity against influenza A virus (H3N2) neuraminidase Con.(nM) 100 10 1 0.1 0.01 Ln (IC₅₀) IC₅₀ Average IC₅₀ NA 1/40 100.43 99.2560.99 13.23 0.63 −0.39549 0.673353 0.542104 NA 1/80 99.89 98.80 63.2024.04 12.33 −0.88951 0.410856 *NA 1/40, NA 1/80 refer to two virusconcentrations used in the experiment.

Example 25 Determination of inhibitory activity of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate against influenza B virus neuraminidase

The inhibitory activity of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid trihydrate against influenza B virus neuraminidase was determinedaccording to the method described in Example 24, wherein influenza Bvirus (B/Jing Fang/76/98) neuraminidase was used in place of influenza Avirus neuraminidase. The results were listed as follows:

Inhibitory activity against influenza B virus (B/Jing Fang/76/98)neuraminidase Con. (nM) 100 10 1 0.1 0.01 Ln (IC₅₀) IC₅₀ Average IC₅₀ NA1/20 97.78 82.01 39.11 15.31 8.81 0.131306 1.140317 1.0691996 NA 1/4096.71 81.45 44.84 19.17 7.92 −0.00192 0.998082 *NA 1/20, NA 1/40 referto two virus concentrations used in the experiment.

Example 26 Experiment of anti-influenza activity of(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid hydrate

A solution was prepared by including 500 pfu (plaque forming unit) ofmice-compliance influenza virus strain FM1 (H1N1) in 50 μl phosphatebuffer (containing 0.42% bovine serum albumin), and then dropped intonasal cavity of mice (BALB/C, female, aged 5-6 weeks, weighted 20 g) toinfect them with an infective dose of 23.7LD₅₀. The compound of theinvention was suspended in physiological saline, and was administered indoses of 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg to the mice byintraperitoneal injection for 5 times, i.e., 1 hour, and 2, 3, 4, 5 daysafter the viral infection, respectively. The assay was performed by 10mice. The result was expressed by the ratio of the number of survivalmice the 14^(th) day after the viral infection to the number of the micein assay. Meanwhile, anhydrous(1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylicacid was used as a control compound.

Dose Reagent Survival number Survival rate blank control physiologicalsaline 10/10  100%  virus control physiological saline 0/10  0% 2.5mg/kg trihydrate 6/10 60% anhydrous 5/10 50% 5.0 mg/kg trihydrate 7/1070% anhydrous 7/10 70%  10 mg/kg trihydrate 8/10 80% anhydrous 7/10 70% 20 mg/kg trihydrate 9/10 90% anhydrous 9/10 90%

What is claimed is:
 1. A method for preparing a compound of the general formula (I),

wherein X is 2.0 or 3.0, comprising the steps of: suspending (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid crude product in water and dissolving by heating to 50-90° C. to afford a first solution; allowing the first solution to stand for a moment and then adding activated carbon to the first solution; refluxing the first solution by heating for 10 minutes and filtering while the first solution is hot to remove the activated carbon; cooling the first solution to 35-75° C. and then adding an organic solvent to the first solution to afford a second solution; adding a crystal seed of the compound of the general formula (I) to the second solution; placing the second solution in a sealed vessel, allowing the second solution to stand, and cooling the second solution at a suitable cooling rate to precipitate the compound of the general formula (I) as a crystal product, and then filtering and collecting the compound of the general formula (I).
 2. The method according to claim 1, wherein the organic solvent is one selected from the group consisting of methanol, ethanol, n-butanol, acetone and acetonitrile or a mixture thereof.
 3. The method according to claim 2, wherein the organic solvent is one selected from the group consisting of methanol and acetone.
 4. The method according to claim 1, wherein the second solution comprises water and the organic solvent in a ratio from 1:5 to 100:1.
 5. The method according to claim 1, wherein the cooling rate is from 0.1° C./min to 5° C./min.
 6. A method for preparing a compound of the general formula (I),

wherein X is 2.0 or 3.0, comprising the steps of: dissolving (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid crude product in water to afford a first solution; adding an organic solvent to the first solution to afford a second solution; cooling the second solution at a suitable cooling rate to precipitate the compound of the general formula (I) as a crystal product; and separating the crystal product from the second solution to isolate the compound of the general formula (I).
 7. The method according to claim 6, wherein the organic solvent is one selected from the group consisting of methanol, ethanol, n-butanol, acetone and acetonitrile or a mixture thereof.
 8. The method according to claim 7, wherein the organic solvent is one selected from the group consisting of methanol and acetone.
 9. The method according to claim 6, wherein the second solution comprises water and the organic solvent in a ratio from 1:5 to 100:1.
 10. The method according to claim 6, wherein the cooling rate is from 0.1° C./min to 5° C./min.
 11. The method according to claim 6, wherein said dissolving (1S,2S,3 S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid crude product in water to afford a first solution comprises: suspending (1S,2S,3 S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid crude product in water to afford a suspension; and heating the suspension to 50-90° C. to dissolve the (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethyl-butyl]-4-guanidino-2-hydroxy-cyclopentyl-1-carboxylic acid crude product to afford the first solution.
 12. The method according to claim 6, further comprising, before said adding an organic solvent to the first solution to afford a second solution: adding activated carbon to the first solution; heating the first solution to reflux; filtering the first solution; and cooling the first solution to 35-75° C.
 13. The method according to claim 6, further comprising, before said cooling the second solution at a suitable cooling rate to precipitate the compound of the general formula (I) as a crystal product, placing the second solution in a sealed vessel.
 14. The method according to claim 6, further comprising, before said cooling the second solution at a suitable cooling rate to precipitate the compound of the general formula (I) as a crystal product, adding a crystal seed of the compound of the general formula (I) to the second solution.
 15. The method according to claim 6, further comprising, before said adding an organic solvent to the first solution to afford a second solution, adjusting the temperature of the first solution to 35-75° C.
 16. The method according to claim 15, wherein the cooling rate is from 0.1° C./min to 5° C./min.
 17. The method according to claim 6, wherein X is 2.0.
 18. The method according to claim 6, wherein X is 3.0.
 19. The method according to claim 6, wherein said cooling the second solution at a suitable cooling rate comprises: cooling the second solution from 75° C. to −10° C. within 6 hours; and allowing the second solution to stand at −10° C. for 12 hours.
 20. The method according to claim 6, wherein said cooling the second solution at a suitable cooling rate comprises allowing the second solution to stand for 12 hours. 